Neurodevelopmental disorders including intellectual disability, autism, juvenile intractable seizures, and schizophrenia have high socioeconomic impact, yet poorly understood etiologies. Recent large scale sequencing efforts identified de novo mutations as a major cause of neurodevelopmental disorders. Most de novo mutations arise in the paternal germline to confer a growth advantage to mutant spermatogonia in what has been termed ?selfish spermatogonial selection?. Protein phosphatase 2 (PP2A), one of the major Ser/Thr phosphatases is a known regulator of growth and differentiation and a suspected tumor suppressor. A trimeric enzyme of catalytic (C), scaffolding (A), and variable regulatory subunits (B,B',B''), PP2A can exist in >50 subunit combinations in mammalian cells, presumably with distinct localization, substrates, and regulatory mechanisms. A surge of de novo mutations in PP2A uncovered since 2015 defined two new classes of autosomal-dominant mental retardation. The most common class is caused by recurrent missense mutations in one of the 12 PP2A regulatory subunit genes, PPP2R5D, the product of which, B'? predominates in human testes and brain. The same de novo PPP2R5D mutations cause human overgrowth, a syndrome commonly associated with intellectual disability and autism. This exploratory proposal seeks to identify molecular mechanisms by which recurrent de novo mutations in PPP2R5D (B'?) cause neurodevelopmental disorders. Because some neurodevelopmental disorders are reversible, our results may lead to new pharmacological interventions. Predicated by our published work predating the discovery of PP2A mutations in mental retardation, we hypothesize that de novo mutations in PPP2R5D cause neurodevelopmental disorders by a novel change-of-function mechanism. Specifically, we suggest that basic amino acids introduced into an acidic substrate-binding surface alter PP2A substrate specifity to impair some and favor other dephosphorylation events. This in turn may enhance growth/proliferative signaling pathways over those that mediate cell cycle exit, differentiation, and morphogenesis. To address this hypothesis, the two aims of this proposal will delineate consensus sequences for dephosphorylation by wild-type and mutant PP2A enzymes, identify their cellular substrates by quantitative phosphoproeomics, and uncover phenotypes in cell models of neuronal development. Fundamental insights from this proposal are expected to pave the way for new patient-derived cell models, animal models, diagnostic tests, as well as ultimately for PP2A-targeted therapies of neurodevelopmental disorders.